Mutants of the factor VII-activating protease and detection methods using specific antibodies

ABSTRACT

Mutants of the DNA sequence coding for the protease (FSAP) which activates blood clotting factor VII and single-chain plasminogen activators, the mutants comprising a G/C base exchange at nucleotide position 1177 and/or a G/A base exchange at nucleotide position 1601, are described. The corresponding protease has a Glu/Gln exchange at amino acid position 393 and/or a Gly/Glu exchange at amino acid position 534. Diagnostic methods which are used for detecting FSAP in body fluids or tissue cells and also for identifying patients with genetic heterozygous or homozygous FSAP expression are also described. In addition, antibodies against FSAP and its mutants are disclosed and diagnostic methods which can be used to detect antibodies against FSAP and its mutants are specified.

This application is a division of Application Ser. No. 09/912,559, filedJul. 26, 2001, now U.S. Pat. No. 6,831,167 and claims priority to GermanApplication Nos. 100 36 641.4, filed Jul. 26, 2000, 100 50 040.4, filedOct. 10, 2000, 100 52 319.6, filed Oct. 21, 2000, and 101 18 706.8,filed Apr. 12, 2001. All of the above applications are incorporated byreference herein.

The invention relates to mutants of the blood clotting factorVII-activating protease (FSAP), to methods for detecting FSAP and itsmutants at the protein level and also RNA/DNA level or in a tissuesample and also to specific antibodies for said detection methods.

The German patent application 199 03 693.4 already discloses a proteasewhich has been isolated from blood plasma and which can activateclotting factor VII. Owing to this first finding, said protease wasdenoted factor VII-activating protease (FSAP). Detailed studies showedthat FSAP is also a potent activator of single-chain plasminogenactivators such as prourokinase or single-chain tissue plasminogenactivator (sct-PA). Owing to these properties, possible applications ofFSAP have been described, for example its application ascoagulation-promoting agent based on FVII activation-assistedacceleration of coagulation. FSAP may also be used, alone or incombination with plasminogen activators, for fibrinolysis, for examplein the case of thrombotic complications.

As described in the German patent applications 199 03 693.4 and 199 26531.3, assays for detecting the protease have been developed which makeit possible to quantify both the FSAP antigen content and its activity,for example in the plasma. In this connection, antigen determination ispreferably carried out by means of an ELISA assay. As described in theGerman patent application 199 26 531.3 FSAP activity can be determinedby quantifying the activation of prourokinase to urokinase and reactionof the latter with a chromogenic substrate by subsequently measuring thedifference in extinction. Surprisingly, it was found during saidactivity assay that the FSAP proenzyme isolated from, for example,plasma was activated under the chosen incubation conditions and thusmade activation of prourokinase possible. More recent studies have shownthat FSAP is converted into the active form by self activation and thus,for example, can activate prourokinase or FVII. This is furthersupported by the abovementioned incubation conditions, i.e. neutral toalkaline pH, calcium ions and heparin. Moreover, most recent resultsindicate that prourokinase/urokinase (or single-chain and double-chaintPA) themselves cause or assist activation of single-chain FSAP.

Using the two abovementioned assay systems, i.e. the ELISA andprourokinase activation assay, more than 180 plasmas of healthy blooddonors were studied. It was found that 5 to 10% of all samples had amarkedly decreased potential of FSAP-effected prourokinase activationcompared with a plasma pool (of more than 100 healthy donors) or withthe average of the whole test group.

In contrast, average FSAP antigen values were measured in the majorityof said donors (with decreased activity). It is assumed therefore thatin the blood samples studied one or more FSAP modifications which wouldlead to reduced or missing activities could be present. The reason forthis could be polymorphisms in the population, i.e. one or moremutations in the FSAP structures, which are indicated in a modificationof the FSAP amino acid sequence, as was already assumed in the Germanpatent application 199 26 531.3. The activities which are usually 50 to70% lower than the average value of all donors studied indicate aheterozygous mutation. The resulting phenotype could be a probably equalpresence of both FSAPs, namely the wild type FSAP and the mutantvariant, in the plasma. Assuming the mutated variant had (almost)completely lost the property to activate prourokinase, then on averageabout half the activity would be measured. In addition, however,pseudohomozygous manifestations of heterozygous mutations of otherproteins have also been described, in which merely the mutated proteinwhich itself, however, had only lost part of the appropriately detectedbiological property was detectable.

In order to exclude that the deficiency or reduction in unknownpotential cofactors is responsible for the detected reduction in FSAPactivity, FSAP samples of three donors whose donated samples hadrepeatedly shown significantly reduced activity were purified. Thehighly purified proteins likewise showed a markedly reduced activitycompared with FSAP purified from the plasma pool. This reduces thepossibility of a cofactor influence and increases that of a proteinmodification in the abovementioned sense. A surprising result was theapparently unreduced potential for activating factor VII. For thisreason, mutants of this kind are particularly suitable for theabovementioned application as clotting-promoting agent, as described inthe German Patent application 199 03 693.4, since their fibrinolyticpotential is apparently limited. Said mutants may be preparedrecombinantly or transgenically based on the findings described below ofthe nucleotide sequence modifications. However, they may, like thecorresponding FSAP protein (single-chain or double-chain FSAP), also beisolated directly from natural sources such as blood plasma. The GermanPatent applications 199 03 693.4, 199 37 21 9.5 and 199 37 21 8.7 havealready described methods which involve preparation of FSAP, preferablywith the aid of immunoabsorption, as is illustrated in detail in theGerman Patent application 100 36 641.4. However, as far as it is known,the monoclonal antibodies used up until now do not discriminate betweenFSAP wild type and FSAP mutants. Accordingly, only monoclonal antibodiesreacting specifically with the mutants can be used for preparing themutants. It is possible to obtain the antibodies by immunization withthe mutant. It is also possible to use peptides with protein regionscorresponding to amino acids 389 to 397 ( . . . SFRVQKIFK . . . ) of SEQID NO:4 and/or 534 to 539 ( . . . EKRPGV . . . ) of SEQ. ID NO:4 forimmunization and for generation of corresponding antibodies according toknown methods. In addition, said antibodies are also used tospecifically detect said mutants, for example as reagents in detectionmethods such as ELISA Western Blots, in immunohistology or influorescence assisted cell sorting (=FACS).

On the other hand, antibodies specific for FSAP wild type or directedagainst the corresponding amino acid sequences of the wild type, forexample, directed against the amino acid sequence, 389 to 397 ( . . .SFRVEKIFK . . . ) of SEQ ID NO:3 and/or against the amino sequence 534to 539 ( . . . GKRPGV . . . ) of SEQ ID NO:3 may be used especially inhumanized form as a pharmaceutical for prophylactic or therapeuticinhibition of FSAP activity in order to counteract, for example,hyperfibrinolyses causing bleedings. In addition, it is also possible touse said antibodies for the purification, detection and distinction ofwild type FSAP in the above described manner.

The genomic FSAP sequence was identified in the gene bank underaccession no. AC 006097 by comparison with the known cDNA sequence(Choi-Miura, Accession No. S 83182) and intron and exon sequences werederived in the process. A total of 12 primer pairs was designed in orderto be able to amplify the coding sequences in specific PCR reactionstogether with a small part of the respective flanking intron sequences.

First genomic DNA from the blood of 2 subjects with reduced activity andfrom four subjects with normal prourokinase activity was isolated,amplified using all primer pairs and then the DNA sequence wasdetermined using the PCR primer. The result is shown in Table 1. A totalof 4 nucleotide positions in the coding region were polymorphic, i.e. atthese positions two bases were detected simultaneously. It can thereforebe assumed that these cases are heterozygous, having one wild type andone mutant allele. Two of these (at positions 183 and 957) are thirdbase exchanges which result in amino acid exchange. The other two whichwere found only DNA of the subjects with reduced prourokinase activitylead to amino changes as depicted in Table 1.

TABLE 1 DNA sequence at nucleotide positions* Subject ProUK No. activity183 957 1177 1601 S83182 T G G G 9689 normal T/C G G G 9690 normal T/C GG G 9704 normal T G/A G G 9706 normal T G/A G G 9714 reduced T G G/C G/A9715 reduced T G G/C G/A *where 1 is A of the start codon Amino acid atposition* Subject ProUK NT*183 NT:957 NT:1177 NT:1601 No. activity AA*61AA:319 AA:393 AA:534 S83182 His Lys Glu Gly 9689 normal His Lys Glu Gly9690 normal His Lys Glu Gly 9704 normal His Lys Glu Gly 9706 normal HisLys Glu Gly 9714 reduced His Lys Glu/Gln Gly/Glu 9715 reduced His LysGlu/Gln Gly/Glu *NT-Nucleotide position; AA-amino acid position

In order to study the correlation of the two mutations with reducedprourokinase activity, the DNAs of further individuals was sequenced atthese positions. The result is summarized in Table 2. All 6 subjectshaving reduced prourokinase activity were heterozygous at the nucleotideposition 1601 (Gly-Glu exchange), and four were additionallyheterozygous at position 1177 (Glu-Gln exchange). None of the 11subjects in total having normal prourokinase activity or prourokinaseactivity in the lower normal range had the abovementionedheterozygosities. This result suggests that at least the exchange inamino acid position 534 is causally linked to reduced prourokinaseactivity. Whether an amino acid exchange at position 393 only can leadto a reduction in prourokinase activity, is still uncertain at themoment.

TABLE 2 DNA sequence at nucleotide position Subject No. ProUK activity1177 1601 9714 Low C/G A/G 9715 Low C/G A/G 9802 Low C/G A/G 10032 Low GA/G 10039 Low C/G A/G 10047 Low G A/G 9698 Lower normal range G G 9702Lower normal range G G 9711 Lower normal range G G 9712 Lower normalrange G G 10038 Lower normal range G G 9689 Normal G G 9690 Normal G G9704 Normal G G 9706 Normal G G 9803 Normal G G 10043 Normal G G

The invention thus relates to a mutant of the DNA sequence coding forthe protease (FSAP) which activates blood clotting factor VII andsingle-chain plasminogen activators, which mutant comprise a G/C baseexchange at nucleotide position 1177 and/or a G/A base exchange atnucleotide position 1601.

The nucleotide sequence SEQ ID NO:1 of the attached sequence listingrepresents the wild type sequence. The DNA sequence of the mutant withthe two exchanges at nucleotide positions 1177 and 1601 is described bySEQ ID NO:2. The corresponding wild type amino acid sequence can befound in SEQ ID NO:3. SEQ ID NO:4 shows the mutant amino acid sequencewith the two amino acid exchanges (Glu-Gln 393 and Gly-Glu 534).

The detection of the DNA and amino acid sequences mentioned in thesequence listing has created the conditions for developing diagnosticmethods for identifying patients having genetic heterozygous orhomozygous FSAP expression. It is possible to detect the mutationseither in the genomic DNA or in the mRNA derived therefrom. However,they can also be detected successfully at their protein level usingmonoclonal or polyclonal antibodies which are directed against themutant having the modified amino acid sequence or against the wild type.

This makes it necessary to produce antibodies suitable for this purposeand thus to develop reliable diagnostic methods and assay systems.

The invention therefore also relates to monoclonal antibodies againstblood clotting factor VII-activating protease and its mutants and alsoto their use for the detection and activity determination of FSAP andits mutants.

Assay systems which make it possible both to quantify the FSAP antigencontent and to determine FSAP activity, such as Western Blots or anenzyme immunoassay (ELISA), are already known per se from the GermanPatent applications 199 03 693.4 and 199 26 531.3.

Said assay systems are based on binding and/or detection of FSAP byspecific monoclonal antibodies. Although immunization of, for example,mice, frequently leads to identification of many positive clones withrespect to the expression of a specific monoclonal antibody, often onlya few of said clones are suitable for the abovementioned tasks. It wastherefore the object to detect specific monoclonal antibodies againstblood clotting factor VII-activating protease which can be effectivelyused both for its purification and for its qualitative and quantitativedetection and for determination of its activity.

We have found that these requirements are met to a high degree bymonoclonal antibodies against blood clotting factor VII-activatingprotease, which are produced by hybridoma cell line DSM ACC2453 andhybridoma cell line DSM ACC2454.

Until recently, FSAP was purified mainly in its activated form.Conventional preparation methods such as column-chromatographictechniques, favor the rapid activation and subsequent inactivation ofthe protease. According to the invention, it is now possible to purifyblood clotting factor VII-activating protease and especially itsproenzyme with high yields by coupling one of the abovementionedantibodies to a support, equilibrating said antibody with a liquidcontaining the protease or its proenzyme and then, after washing,obtaining the protease or its proenzyme by elution.

Moreover, the mentioned antibodies are also suitable for detecting bloodclotting factor VII-activating protease or its proenzyme using animmunoassay in which

-   a) a sample which should contain the protease or its proenzyme is    incubated with a first antibody of the invention, fixed to a solid    support, and then, after washing out, a second labelled antibody of    the invention is added and, after washing out again, the signal    produced by the second antibody or other monoclonal or polyclonal    antibodies is measured; or-   b) the protease or its proenzyme contained in the sample to be    tested is fixed to a support, for example by a polyclonal antibody    against the protease or its proenzyme and is detected with a    labelled inventive antibody alone or in a mixture with an unlabelled    inventive antibody and subsequent detection of the monoclonal    antibody or-   c) a sample to be tested for the protease or its proenzyme is added    to an inventive antibody fixed to a support in the presence of a    labelled protease or its proenzyme and the signal produced by the    label is measured.

Another possibility for detecting blood clotting factor VII-activatingprotease or its proenzyme is to carry out the detection using theWestern Blot method by an immunological reaction with a labelledinventive antibody alone or in a mixture with an unlabelled inventiveantibody and subsequent detection of the monoclonal antibody, forexample by labelled protein A or G or a labelled monoclonal orpolyclonal antibody directed against the monoclonal antibody.

Finally it is also possible to determine the activity of blood clottingfactor VII-activating protease or its proenzyme in protein solutions byincubating the protein solution containing the protease and/or itsproenzyme to a solid support to which an anti-protease monoclonalinventive antibody has been coupled beforehand and, after washing outthe solid support, incubating the protease and its proenzyme fixed tosaid solid support with reagents allowing determination of theiractivity. The activity of the protease or its proenzyme can then bemeasured by photometric determination of the extinction appearing due tothe action on chromogenic substrates.

Other possibilities for determining the activity of the protease or itsproenzyme are to measure

-   -   its action of inactivating blood clotting factors VIII/VIIIa or        V/Va or    -   its action of shortening blood clotting times in global clotting        assays or    -   its action of activating plasminogen activators or    -   its action of activating blood clotting factor VII.

Both the complete monoclonal antibodies and their fragments such asF(ab′)₂ or Fab are suitable for application in the abovementionedmethods. After labeling with a radioactive or fluorescent orenzymatically active substance, the antibodies or their fragments areemployed as detecting aids in an immunoassay or in a Western Blotdetection method. The unlabelled monoclonal antibodies or fragments maylikewise be employed, but then detection or immobilization is carriedout, for example, by a labeled antibody, directed against the mouseantibody, or its labelled fragment (sandwich method). The inventivemonoclonal antibodies or their fragments may be employed alone or asmixtures. This is particularly recommended for Western Blots, since SDSgel electrophoreses are frequently carried out under reducing conditionsof the samples. Since the two FSAP polypeptide chains are linked to oneanother merely via a disulfide bridge, the molecule disintegrates duringreduction into two chains, namely the heavy and the light chain, theformer being recognized by the monoclonal antibody of DSM ACC2454, andthe latter by the monoclonal antibody of DSM ACC2453. Thus, for thedetection of both chains the two inventive monoclonal antibodies ortheir fragments are required.

The monoclonal antibodies according to the invention were prepared andcharacterized as follows:

Immunization

Three female balb/c mice (approx. 6 weeks old) were immunized with FVIIIactivator. The first injection consisted of 0.2 ml of the antigen (10μg) mixed with 0.2 ml of complete Freund's adjuvant. In the threefollowing boost injections (each 2 weeks apart) the antigen (20 μg in0.2 ml) was administered without adjuvant (all injections i.p.). Theimmunogen was diluted in PBS. After the last injection, the serum titerwas determined by means of indirect ELISA by coating a microtiter platewith FVII activator. The mouse with the highest serum titer was selectedfor the fusion.

Fusion

About three weeks after the last application, the antigen wasadministered on three successive days (10 μg in 0.1 ml i.v.). On thenext day (day 4) the mouse was sacrificed after taking blood. The spleenwas removed and the spleen cells were isolated. The spleen cells werethen fused with the murine myeloma cell line SP2/0-Ag 14. The fusionreagent was polyethylene glycol 4000 (Merck). The fusion was carried outusing a modification of the original Kbhler/Milstein method. The cellswere distributed on 24-well culture plates. The medium used was Dulbeccomod. Eagle's medium with 10% fetal calf serum and HAT for selection.After about two weeks, the cell clones grown were transferred to thewells of a 48 well plate and coded.

Hybridoma Screening

The culture supernatant was taken from 1728 grown clones and assayed bymeans of ELISA for the presence of mouse IgG. With the aid ofimmobilized FVII activator, mouse IgG-positive supernatants were testedfor specificity (ELISA). Of the cell lines assayed, 108 cell lines wereidentified as specific for FVII activator and stored in the frozenstate.

The two hybridoma cell lines denoted DSM ACC2453 and DSM ACC2454 wereselected for further studies. The specificity of the antibodies producedby said cell lines was confirmed by BIACORE and binding kinetics wasdetermined. The two monoclonal antibodies are of the IgG1 type.

With the aid of the described antibodies against FSAP wild type andagainst its mutants it is possible to carry out diagnostic methods fordetecting the mutants by

-   a) incubating a sample which could contain the mutant with a first    antibody as claimed in claim 7, fixed to a solid support, then,    after washing, adding a second, labelled antibody as claimed in    claim 7 or a labelled antibody directed against the wild type and,    after washing out again, measuring the signal produced by the second    antibody, or-   b) incubating a sample which could contain the mutant with a first    antibody fixed to a solid support and directed against the wild    type, then, after washing, adding a second, labelled antibody as    claimed in claim 7 and, after washing out again, measuring the    signal produced by the second antibody, or-   c) fixing the sample to be tested for the presence of the mutant to    a support and detecting said sample with a labelled antibody as    claimed in claim 7 alone or in a mixture with an unlabelled antibody    and subsequent detection of the labelled antibody, or-   d) adding a sample to be tested for the presence of the mutant to an    antibody as claimed in claim 7, fixed to a support, in the presence    of a labelled mutant and measuring the signal produced by the label.

Preference is given to a diagnostic method in which the FSAP activity ismeasured by incubating the protease-containing sample to a solid supportto which an anti-protease antibody as claimed in claim 7 has beencoupled beforehand and, after washing out the solid support, incubatingthe protease fixed to said support with reagents which allowdetermination of its activity.

In this connection, the protease activity can be measured by photometricdetermination of the extinction appearing following the action onchromogenic substrates.

It is also possible to determine the protease activity by measuring

-   -   its action of inactivating blood clotting factors VIII/VIIIa or        V/Va or    -   its action of shortening blood clotting times in global clotting        assays or    -   its action of activating plasminogen activators or its action of        activating blood clotting factor VII.

Finally, there are also methods available in which the action ofactivating plasminogen activators is measured, by activating the

-   -   single-chain urokinase (scuPA, single chain urokinase        plasminogen activator) or the    -   single-chain-tPA (sctPA, single chain tissue plasminogen        activator).

The mutations responsible for the reduction of prourokinase activity canbe detected at the DNA and RNA level by using methods as they are alsoapplied for detecting single nucleotide polymorphisms, for example

-   -   the cDNA amplification of the RNA or amplification of the        genomic DNA and their subsequent sequencing;    -   the detection of the mutation at the cDNA level or genomic DNA        level or their amplification by    -   hybridization with sequence-specific probes which may also carry        labels for the detection, such as enzymes, alkaline phosphatase,        HRP and their substrates, fluorescent dyes, also        reporter-quencher pairs (such as, for example, scorpions,        molecular beacons, TaqMan probes), radioisotopes, chromophores,        chemiluminescence labels and electrochemiluminescence labels) or        -   by methods such as selective 2′-amine acylation,            electrochemical oxidation of nucleic acids by “minor groove            binder” oligonucleotide conjugates or by HPLC.

On the basis of the test results which were obtained by theabovementioned antigen assays and activity assays it was possible tostudy three groups of healthy donors regarding potential mutations atthe genomic level. For this purpose, blood was taken from the donors andthe blood cells were separated from the plasma by centrifugation. Theplasmas were then used to quantify the FSAP antigen and activity levelsand were divided according to the latter into three groups, namely into“high/average”, “average/reduced” and “significantly reduced”. The bloodcells obtained were then used to extract DNA/RNA and the resultsdepicted on page 5 and in table 1 were determined therefrom.

Based on the present results, it is now possible to detect rapidly oneor both of the mutants described, no matter whether their genotype isheterozygous or homozygous, at the level of the corresponding FSAPnucleotide sequence. Whereas the abovementioned antigen and activityassays reflected quite well the genotype in a healthy donor, this canbecome difficult or impossible when the FSAP plasma levels areinfluenced. Thus, parameters such as hormonal fluctuations, lifestyle,etc., but particularly pathological conditions, more or less stronglyinfluence antigen and/or activity levels. As described in the GermanPatent application 199 26 531.3, the measurable FSAP activity during aheart attack can increase markedly compared with the normal value withscarcely increased antigen content, and, as a result, donors which havea reduced FSAP activity when healthy, now appear to be “average”.

For example, studies on whether patients with FSAP mutation run anincreased risk of suffering thrombotic complications such as heartattacks are possible only with difficulty, owing to the abovementionedrestrictions. On the other hand, for example, liver failures may lead toreduced plasma levels, and this likewise may lead to misinterpretationsof the “true” genetic predisposition. In contrast, an FSAP mutationassay at the DNA/RNA level is independent of temporary events. Thecombination of all of the assays mentioned allows a complete picture ofthe donor/patient, i.e. the evaluation of a potential mutation and ofthe acute state regarding an influence on the antigen-activity ratio.This may result in prophylactic and therapeutic measures.

As described above, exclusively heterozygous blood donors whose bloodplasma contains normal FSAP at about 50% and the FSAP mutant at about50% have been found up until now. This results in an about 50% reducedactivity level of plasmas in which both types of FSAP molecules arepresent. Plasma pools which have been obtained from the blood of 100 andmore donors therefore also contain 5 to 10% of FSAP mutants, dependingon the population. This results in a corresponding probability toreceive in blood transfusions donor blood plasma which contains the FSAPmutant. If blood containing this mutant is administered to a recipientwho cannot produce said mutant, then said mutant may be recognized asextraneous and appropriate antibodies can be generated. Subsequentadministration of the FSAP mutant at a later stage may lead toimmunological reactions in the recipient the side effects of which arefamiliar to the skilled worker.

Conversely, in a homozygous blood recipient who produces merely the FSAPmutant but not normal FSAP, the latter is recognized as “extraneous” andthe appropriate antibodies against it are produced.

FSAP affects hemostasis and the cellular processes connected therewith.By involvement in blood clotting and/or fibrinolysis, it also affectsthe wound healing reaction. Moreover, FSAP, due to its property ofhaving a high affinity to glycosaminoglycans, can bind to cells andother matrices and therefore is probably physiologically andpathophysiologically involved in cell migration and cellular-proteolyticprocesses.

Anti-FSAP antibodies thus may influence all FSAP-mediated activities. Inthe case of autoantibodies against FSAP appearing, it is, in addition toan impairment of the physiological functions, possible thatimmunocomplexes (FSAP+antibody) contribute to side effects of knownautoimmune diseases. This may lead, for example locally in theendothelium, to vasculitides. Neutralization of FSAP activity asprofibrinolytic agent could also contribute to a thrombosis-promotingstate.

There is, therefore, the need for a diagnostic method for detecting theabove described antibodies.

The invention therefore also relates to a diagnostic method fordetecting antibodies against factor VII-activating protease (=FSAP)and/or against an FSAP mutant formed by the exchange of one or moreamino acids, which method comprises letting a sample which could containantibodies react with the FSAP and/or FSAP mutant which are fixed to asolid support, incubating, after washing, the antibody bound to theprotease(s) with a labelled human anti-immunoglobulin or a labelledprotein A and determining the signal emitted by the bound labelledsubstance.

The diagnostic method of the invention is expediently carried out usingthe ELISA technique in which FSAP and/or the FSAP mutant are bound to amatrix, for example to a microtiter plate. For optimal presentation ofFSAP and/or FSAP mutant, the plate may be coated beforehand withmonoclonal or polyclonal antibodies or their F(ab′)₂ or Fab fragmentsand then loading said plate with FSAP and/or the FSAP mutant. Since FSAPand the mutant bind very well to dextran sulfate, heparin and similarsubstances, the prior coating with said agents for FSAP binding is alsopossible. After washing, the support or the microtiter plate is, whereappropriate, in addition blocked and washed using the agents known forthis purpose, such as detergent or albumin, and then incubated with thesolution to be assayed. FSAP antibody-containing solutions may be bloodserum, plasma and other body fluids such as synovial fluids, CSF,sputum, tears or seminal plasma or else cell lysates.

After incubating and washing the support, a suitable detection agent isthen used. The assay substances necessary for detecting the variousantibody classes such as IgG, IgM, IgA, IgE and the subclasses belongingthereto are commercially available as labelled reagents. The antibodytiter may be detected and quantified by a photometric determinationmeasuring the extinction which is caused by cleavage of a chromogenicsubstrate by an enzyme coupled to the anti-human antibody. However, itis also possible to measure fluorescence which is emitted by afluorescent group linked to the antibody used for detection. Finally, itis also possible to carry out the detection using radiometricmeasurement, if the substance used for detection is labelled with aradioactive group.

The determination of antibodies against FSAP and/or in particular FSAPmutants makes it possible to identify the risk involved in a bloodtransfusion prior to carrying out said blood transfusion and to avoiddangerous complications by suitable measures.

The invention further relates to a diagnostic method for theimmunohistochemical detection of the blood clotting factorVII-activating protease, its proenzyme or its mutants or fragments,which method comprises letting an anti-protease, labelled, monoclonal orpolyclonal antibody or one of its fragments react with a tissue sample,and washing out the unbound antibody or its fragments and determiningthe signal emitted from the bound antibody or one of its fragments.

The method may also be carried out by letting an unlabelled monoclonalor polyclonal antibody, directed against the protease, its proenzyme ormutants or fragments thereof, or one of its fragments react with thetissue sample, washing out the unbound antibody orits fragments, thenletting a labelled anti-antibody react with the tissue and, afterwashing out the unbound labelled anti-antibody, determining the signalemitted from the bound anti-antibody or its fragments.

It was also found that monoclonal or polyclonal antibodies directedagainst FSAP are very well suited to detecting FSAP in tissue sectionsof human origin, when said antibodies are labelled with chromophoric orluminescent groups. Anti-FSAP polyclonal antibodies obtained byimmunization of rabbits, sheep, goats or other mammals are suitable forsaid detection as well as monoclonal antibodies. Particularly suitablefor the histological specific detection of FSAP which may be presentboth in the active form and in the proenzyme form or as fragment are themonoclonal antibodies of hybridoma cell lines DSM ACC 2453 and DSM ACC2454. Complexes of activated FSAP with inhibitors such as antiplasminmay also be detected in this way. Suitable for this purpose are allcommon histological detection methods such as light microscopy,fluorescence microscopy and electron microscopy.

Suitable for detecting FSAP in the abovementioned methods are both thecomplete polyclonal and monoclonal antibodies and their fragments suchas F(ab′)₂ or Fab, as long as they are labelled with a detectable group.The abovementioned antibodies or their fragments may be applied alone oras a mixture. This is particularly recommended in case one of therecognized epitopes is obscured. For example, a protein domain may notbe accessible for an antibody due to cellular association, but is boundby another antibody having specificity for a different FSAP region.Antibodies which are directed against human FSAP, the wild type and/oragainst mutants thereof and which are described in more detail in theGerman Patent application 100 52 319.6 may also be employed fordetection of FSAP in tissue sections of human origin.

The findings obtained so far on the immunohistochemical detection ofFSAP can be summarized as follows:

-   -   FSAP is detected in almost all of the human tissues studied up        until now;    -   endocronologically active cells such as Leydig cells or the        endocronologically active cells of the islets of Langerhans of        the pancreas can be very strongly stained intracytoplasmatically        using antibodies carrying chromophoric groups;    -   epithelia and endothelia display according to their location a        more or less strong intracytoplasmatic immunoreaction with        antibodies against FSAP;    -   gangliocytes and dendrites of the cortex display high        concentrations of FSAP, and this is detected by a strong        immunohistological color reaction with chromophoric antibodies;    -   plasma cells display an intensive intracytoplasmatic coloration        with chromophoric antibodies;    -   mesenchymal stroma cells display in complex tissues only a weak        or no color reaction toward FSAP.

FSAP is thus a protein which can be regarded as a normal cellconstituent. So far FSAP was found located both intracellularly andextracellularly, with the former compartment being markedly morestainable. The inventive detection of FSAP by the mentioned antibodiesor their fragments makes it possible to identify the followingpathological processes:

-   -   endocrinologically active tumors and neuro-endocrine tumors;    -   angiogenic endothelia and endothelia of the capillary        endothelium; and also    -   angiogenically active tumors such as gliomas and glioblastomas,        but also, for example, vascular tumors such as        hemangioendothelioma or hemangiopericytoma and angiosarcoma;    -   wound healing reactions, granulation tissue and collagenoses;    -   arteriosclerotic, (micro)thrombosed and necrotic areas;    -   neurodegenerative disorders such as Alzheimer's disease,        Parkinson's disease or as spongiform encephalitides, for example        caused by prion proteins;    -   gammopathies and myelomas.

FSAP is detected by using preferably monoclonal antibodies of hybridomacell lines DSM ACC 2453 or DSM ACC 2454.

The diagnostic method of the invention is illustrated in more detail bythe following example:

The immunohistochemical reactivity of the FSAP-specific monoclonalantibodies of hybridoma cell lines DSM ACC 2453 and DSM ACC 2454 wasstudied by preparing from adult human tissue and malignant urologicaltumors 10 μm thick paraffin sections and subsequently dewaxing saidsections which were treated in citrate buffer in the microwave for 3times 5 minutes. First, an unlabelled antibody of the abovementionedhybridoma cell lines was allowed to react with said sections for 30minutes. After washing out the tissue section, a labelled anti-mousedetection antibody was allowed to react with the tissue likewise for 30minutes and then the bound FSAP antibody was made visible by forming theAPAAP complex (Alkaline Phosphatase/Anti-Alkaline Phosphatase complex)and by staining with chromogen and counterstaining with hemalum.

As a negative control, each tissue was separately incubated with thedetection antibody—without prior incubation with the FSAP antibody—inorder to make potential unspecific reactions of the detection visible.In addition an antibody against α-keratin was included as a positivecontrol.

The results of the immunohistochemical study of normal human tissue aresummarized in Table 1:

Antibodies against FSAP, clone DSMZ ACC2454 and DSMZ ACC 2453 Humannormal tissue DSMZ DSMZ ACC2454 ACC2453 Esophagus Squamous 2+ 2+epithelium Secretory units 0 0 Acinar ducts 2+ 2+ Musculature 1+ 1+Stroma 1+ 1+-2+ Vascular 2+ 2+ endothelium Cardia (stomach) Foveolar 0 0epithelium Glandulae 1+ 2+ cardiacae Mucous 0 0 secretory units Oxynticglands 3+ 3+ Musculature 1+ 1+ Vascular 1+ 1+ endothelium Corpus(stomach) Foveolar 0 0-1+ epithelium Corpus gland 2+ 2+ body Musculature0 1+ Vascular 1+ 1+ endothelium Duodenum Epithelia 0-1+ 1+ Brunner's 0 0glands Musculature 0 0-1+ Lymphatic 1+ 2+ follicle Ganglion cells 2+ 3+Vascular 1+ 1+ endothelium Small intestine Epithelia 2+ 3+ Musculature1+ 1+ Stroma 1+ 2+ Ganglion cells 3+ 3+ Vascular 1+ 1+ endotheliumColon/Rectum Epithelia 1+ 1+ Lymphatic 1+ 1+ follicles Plasma cells 1+1+ Vascular 1+ 0 endothelium Epididymis Epididymis duct 2+ 2+ Efferent2+ 2+ ductulus Stroma 1+ 1+ Vascular 1+ 1+ endothelium Seminal glandEpithelium 2+ 3+ Musculature 1+ 1+ Vascular 2+ 2+ endothelium Deferentduct Epithelium 2+ 3+ Longitudinal 0 +/− muscle layer Annular muscle 2+3+ layer Vascular 2+ 3+ endothelium Prostate Glandular 2+ 2+ epitheliumMusculature 1+ 1+ Vascular 1+-2+ 1+-2+ endothelium Kidney Tubules 2+ 1+Glomerules 0 0 Medullary 1+ 1+ epithelium Vascular 0-1+ 0-1+ endotheliumBladder Urothelium 2+ 1+-2+ Musculature 2+ 1+ Plasma cells 2+ 2+Fibroblasts 1+-2+ 1+-2+ Peripheral 0 nerve Adrenal gland Glomerular zone2+ 1+ Fascicular zone 1+-2+ (1+) Reticular zone 3+ (1+) Medulla 0 0Vascular 1+ (1+) endothelium Endometrium Glandular 3+ 2+ epitheliumStroma cells 0 1+ Myometrium 1+ 1+ Vascular 1+ 2+ endothelium LungBronchial 2+ 1+ epithelium Alveolar 1+-2+ 1+ epithelium Bronchial 1+ 1+glands Cartilage 3+ 1+ Musculature 1+ 1+ Alveolar 2+ 2+ macrophagesElastic fibers 2+-3+ 2+-3+ Vascular 1+ 1+ endothelium Skeletal 2+ 1+muscles Appendix Epithelia 1+ 3+ Musculature 1+ 1+ Lymphatic follicle 1+2+ Plasma cells 2+ 3+ Vascular 1+ 1+ endothelium Pancreas Epithelia 1+2+ Islets of 3+ 1+ Langerhans Duct epithelium 2+ 2+ Vascular 1+ 1+endothelium Salivary gland Mucous end units 0 0 Serous end units 1+1+-2+ Acinar ducts 1+ 1+ Striate ducts 1+ 1+ Vascular 0-1+ 0-1+endothelium Liver Hepatocytes 2+ 2+ Bile ducts 0 0 Vascular 1+ (1+)endothelium Gall bladder Epithelia 1+ 1+ Musculature 2+ 1+ Vascular 2+1+ endothelium Cystic duct Epithelium 3+ 3+ Musculature 2+ 1+ Ganglioncells 3+ 3+ Vascular 2+ 2+ endothelium Testis Leydig cells 3+ 1+ Sertolicells 1+-2+ 1+ Germ cells 1+-2+ 1+ Vascular 1+ 1+ endothelium Retetestis Epithelium 2+ 2+ Placenta Chorionic 3+ 2+ epithelium Amnioticepithelium 2+ 2+ Decidual cells 2+-3+ 2+ Stroma cells 0 +/− Vascular 1+1+ endothelium Fetal membranes Amniotic epithelium 3+ 2+ Decidual cells3+ 1+ Fibroblasts 3+ 1+-2+ Cervix uteri Glandular 0 0 epitheliumVascular 1+ 0-1 endothelium Stroma 1+ 0-1 Fallopian tube Epithelium 2+3+ Musculature 0 1+ Vascular 1+ 2+ endothelium Breast Epithelia mammary2+ 2+ gland lobules Duct epithelium 2+ 2+ secretory ducts Fibroblasts 01+ Plasma cells 2+ 2+ Vascular 1+ 0 endothelium Thyroid Follicular 2+1+-2+ epithelium Stroma 1+ 1+ Vascular 1+ 0 endothelium Thymus Hassall'sbodies 2+-3+ 2+ Follicles 1+ 2+ Mantle zone (1+) (1+) Starry sky 1+ 1+macrophages Spleen + + Tonsils +/− +/− Lymph nodes +/− +/− Maxillarysinus Respiratory 2+ 2+ epithelium Plasma cells 3+ 3+ Vascular 1+ 1+endothelium Fatty tissue 2+ 2+ Vascular 2+ 2+ endothelium Skin Epidermis2+ 1+-2+ Dermis (1+) 0 Hypodermis (1+) 0 Sweat glands 1+ 0 Vascular 1+ 0endothelium Endocardium 0 0 Fibroblasts 2+-3+ 2+-3+ 0 = negative 1+ =weakly positive 2+ = moderately strong positive 3+ = strongly positive

Endocrine cells such as the islets of Langerhans of the pancreas, theLeydig cells of the testicular interstitium, the decidual cells of theplacenta and the oxyntic gland body of the stomach cardia and also thehighly cylindrical epithelium of the cystic duct display a strongreaction which in part shows fine granules. Strongly positive reactionswere observed in plasma cells located in tissue structures andganglionic cells and nerve cells of the cortex. The decidual cells, theamniotic epithelium and the fibroblasts of fetal membranes displayedvery strong immunohistological stainability as did the epithelium liningthe seminal glands and the enterocytes of the small intestine.

Studies of formalin-fixed, paraffin-embedded tumor material ofurological tumors displayed a weak to moderately strongintracytoplasmatical reaction of different differentiatedadenocarcinomas of the prostate. Tumor cells of seminomatous testiculartumors showed only a weak intracytoplasmatic reaction whilenon-seminomatous tumors (embryonic carcinomas and chorionic carcinomas)had a widely increased stainability of the tumor cells, indicatingincreased concentrations of FSAP.

The diagnostic method of the invention thus allows animmunohistochemical detection of pathological processes in a widevariety of organs.

1. A method for detecting a Factor VII Activating Protease (FSAP)mutation in a human individual, comprising incubating a sample takenfrom said individual with an antibody specifically directed against anepitope comprising a human FSAP amino acid sequence comprising a Glu toGln exchange at amino acid position 393 and/or a Gly to Glu exchange atamino acid position 534, said amino acid sequence positions definedaccording to the FSAP proenzyme amino acid sequence of SEQ ID NO:4; anddetecting the mutation based on antibody recognition.
 2. The method asclaimed in claim 1, which comprises incubating the sample with a firstantibody that binds to FSAP and is fixed to a solid support washing thesolid support; adding a second, labeled antibody, wherein the secondantibody is specific for the epitope of claim
 1. 3. The method asclaimed in claim 1, further comprising measuring FSAP prourokinaseactivating activity in the sample, wherein a measured prourokinaseactivating activity which is lower than that of a normal control sampleindicates an FSAP mutation.
 4. The method as claimed in claim 1, whereinthe method comprises one of Western Blots, immunohistology, orfluorescence-assisted cell sorting (FACS).
 5. The method of claim 1,comprising fixing the sample to a support; incubating said sample with alabeled antibody alone or in a mixture with an unlabelled antibody, anddetecting the labeled antibody, wherein said labeled antibody isspecific for the human FSAP amino acid sequence comprising a Glu to Glnexchange at amino acid position 393 and/or a Gly to Glu exchange atamino acid position 534, said amino acid sequence positions definedaccording to the FSAP proenzyme amino acid sequence of SEQ ID NO:4. 6.The method of claim 1, wherein the antibody is fixed to a solid support,and incubating the support with the sample in the presence of a labeledpolypeptide comprising a human FSAP amino acid sequence comprising a Gluto Gln exchange at amino acid position 393 and/or a Gly to Glu exchangeat amino acid position 534, said amino acid sequence positions definedaccording to the FSAP proenzyme amino acid sequence of SEQ ID NO:4;washing the solid support; measuring the signal produced by the labelbound to the solid support; and detecting the mutation based oninhibition of the binding of label to the solid support.
 7. The methodof claim 3, wherein the FSAP activity is measured by: a) incubating thesample on a solid support to which an antibody has been coupled, whereinsaid antibody is specifically directed against an epitope comprising ahuman FSAP amino acid sequence comprising a Glu to Gln exchange at aminoacid position 393 and/or a Gly to Glu exchange at amino acid position534, said amino acid sequence positions defined according to the FSAPproenzyme amino acid sequence of SEQ ID NO:4; b) washing the solidsupport; and c) incubating the protein fixed to said support withreagents which allow determination of prourokinase activity.
 8. Themethod of claim 1, wherein the antibody is specifically directed againstan FSAP mutant epitope comprising the amino acid sequence SFRVQKIFK,corresponding to amino acids 389 to 397 of SEQ ID NO:4 and/or the aminoacid sequence EKRPGV, corresponding to amino acids 534 to 539 of SEQ IDNO:4, and detecting the mutant epitope based on antibody recognition. 9.The method of claim 4, wherein the sample is a tissue sample and themethod comprises immunohistology.
 10. The method of claim 9, wherein thetissue sample is taken from an endocrine organ.
 11. The method of claim1, wherein the antibody does not recognize an naturally occurring FSAPsequence which does not comprise said exchange at amino position 393 anddoes not comprise said exchange at amino acid position
 534. 12. Themethod of claim 1, which comprises incubating the sample with a firstantibody fixed to a solid support, wherein the antibody is specific foran epitope of claim 1; washing the solid support; adding a secondlabeled antibody that binds to FSAP; washing the solid support; andmeasuring the signal produced by the second antibody.
 13. A method forpurifying a human FSAP mutant polypeptide comprising SEQ ID NO:4, or afragment thereof which comprises a Glu to Gln exchange at amino acidposition 393 and/or a Gly to Glu exchange at amino acid position 534,which comprises fixing one or more antibodies to a support, wherein theantibodies are specifically directed against an epitope comprising ahuman FSAP sequence comprising a Glu to Gln exchange at amino acidposition 393 and/or a Gly to Glu exchange at amino acid position 534,said amino acid sequence positions defined according to the FSAPproenzyme amino acid sequence of SEQ ID NO:4; incubating the supportwith a sample containing the polypeptide; washing the support; andobtaining the polypeptide by elution.
 14. The method as claimed in claim13, wherein the FSAP mutant polypeptide is expressed by recombinantand/or transgenic expression.
 15. The method as claimed in claim 13,wherein the polypeptide is obtained from a source selected from bodyfluids, cell culture supernatants, and fluids of transgenic animals. 16.The method of claim 13, wherein the antibody is specifically directedagainst an FSAP mutant epitope comprising the amino acid sequenceSFRVQKIFK, corresponding to amino acids 389 to 397 of SEQ ID NO:4 and/orthe amino acid sequence EKRPGV, corresponding to amino acids 534 to 539of SEQ ID NO:4; incubating the support with a sample containing thepolypeptide; washing the support; and obtaining the polypeptide byelution.
 17. A method for detecting an FSAP mutation in a humanindividual, comprising incubating a sample taken from said individual orFSAP isolated from the sample with an antibody that specificallyrecognizes a human FSAP amino acid sequence; and determining whether theFSAP of the sample comprises a human FSAP amino acid sequence comprisinga Glu to Gln exchange at amino acid position 393 and/or a Gly to Gluexchange at amino acid position 534, said amino acid sequence positionsdefined according to the FSAP proenzyme amino acid sequence of SEQ IDNO:4.
 18. The method of claim 17, further comprising measuring FSAPprourokinase activating activity in the sample, wherein a measuredprourokinase activating activity which is lower than that of a normalcontrol sample indicates an FSAP mutation.
 19. The method of claim 17,wherein the antibody comprises a monoclonal antibody or antibodyfragment produced from one or both of hybridoma cell lines DSM ACC 2453and DSM ACC 2454.